search

OpenMS / Tutorials

Tutorials as used in the user meetings. (Please assign a git tag for the revision used for the UM)
BSD 3-Clause "New" or "Revised" License
13 stars 19 forks source link

Workflow for isobaric analysis (untested) #166

Closed oliveralka closed 4 years ago

oliveralka commented 5 years ago

Reduced rt setting in IDMapper TMT6-plex (K,N-term) in MSGFPlus The correction Matrix for the TMT experiment has to be set by the user!

jpfeuffer commented 5 years ago

Very nice. I didn't have a look yet (will today). But:

jpfeuffer commented 5 years ago

I will write a TODO list for PRs here.

jpfeuffer commented 5 years ago

Ah this was just a change in one of the WFs but still, maybe you could add the abovementioned points. We should do it for every WF here anyway soon.

oliveralka commented 5 years ago

Did you make the input files accept relative paths inside the workspace?

Should we ship the input data with the workflow?

Did you add a description of the workflow in the workflow settings for easy upload to the KNIME Hub?

Can you please provide a reference / or example on how to do that?

jpfeuffer commented 5 years ago

No, otherwise it gets too big. Data goes here (assume the workspace root is this folder, i.e. specify paths relative to that root): https://abibuilder.informatik.uni-tuebingen.de/archive/openms/Tutorials/Data/

Instructions for description is here: https://docs.google.com/document/d/1uwVCz-4LH7VUzhcsE5FVyrBbRmzDnGQvdFF2ZsA8BYU/edit#heading=h.3hm698qmdoi3

jpfeuffer commented 5 years ago

The workflows have to run from the commandline. You can check by running the shell script executeBatch with arguments: 1) knime executable 2) workspace root

oliveralka commented 5 years ago

There are some errors in the workflow which need to be fixed first - I will update the PR, when it is ready.

oliveralka commented 5 years ago

We have to check if the IDMapper is switched to "precursor" see https://github.com/OpenMS/OpenMS/issues/4216

pavel-shliaha commented 4 years ago

will the pipeline work for MS3 SPS?

oliveralka commented 4 years ago

The IsobaricAnalyzer should use MS3 automatically if possible (@cbielow). Please use the current nightly build of OpenMS in KNIME to have full support.

oliveralka commented 4 years ago

@timosachsenberg @jpfeuffer Added a rought draft for integration into the tutorial. Updated the workflow using epifany - runable - but I still have to check the results. It would be great if you could take a look and make some comments and suggestions, also in terms of explanation of the experimental design for TMT.

Julianus, could you have a peek at the parameters used for epifany?

pavel-shliaha commented 4 years ago

@oliveralka could you please point to where the tutorial for MS3 TMT is and the actual workflow that you recommend?

pavel-shliaha commented 4 years ago

@oliveralka. I have found the workflow, but I cannot find the example files (mzMLs and also the MSstatsTMT table). Could you perhaps give a direct link to it?

oliveralka commented 4 years ago

@pavel-shliaha The small example can be found here: https://abibuilder.informatik.uni-tuebingen.de/archive/openms/Tutorials/Data/latest/Example_Data/IsobaricLabellingTMT/

pavel-shliaha commented 4 years ago

@oliveralka. I downloaded the newest KNIME and OpenMS, the dataset and the database and set file locations on my computer. MSGFPlusAdapter and IsobaricAnalyzer nodes failed. Do they work in your hands?

oliveralka commented 4 years ago

@pavel-shliaha Everything works in my case: KNIME 4.1.1 OpenMS 2.5.0.20200201806

What are the error messages? Can you run the tools in debug mode?

pavel-shliaha commented 4 years ago

@oliveralka. I tried to execute the workflow on two separate computers. On both of them:

KNIME is 4.1.2 OpenMS is 2.5.0.202002241222

How can I run the software in debug mode (googled the question but could not find an answer)? Also if you are interested I can provide access to my computer so you can have a look.

jpfeuffer commented 4 years ago

What does the Console log in the lower right of KNIME say?

pavel-shliaha commented 4 years ago

@jpfeuffer it says I might be missing some of the requirements (trying to isntall them now). I appologise for this but I used the website https://www.openms.de/download/knime-plugin/ and it does not mention the prerequisites. I will now install proteowizard as well

jpfeuffer commented 4 years ago

ok we will update the documentation. But also check the other tab of the lower right view called "Console". And if you need to, you can also check single tools by right-clicking the node and selecting View Standard Output/Error.

pavel-shliaha commented 4 years ago

When I load the workflow I get the following message:

error

I installed all the prerequisites from the suggested link but it did not help

jpfeuffer commented 4 years ago

that can usually be ignored.

pavel-shliaha commented 4 years ago

I installed the prerequisites and the prerequisites for visual studio under this link

https://support.microsoft.com/en-us/help/2977003/the-latest-supported-visual-c-downloads

but it did not help

jpfeuffer commented 4 years ago

As long as it just shows "... FileConverter requirements" this can also be ignored. You just might not be able to use Raw file conversion inside KNIME. We just check registry keys here. Sometimes it does not work correctly.

pavel-shliaha commented 4 years ago

it still fails to execute the workflow

jpfeuffer commented 4 years ago

I thought so. Without the requirements no OpenMS node would work. That is why you need to check the Console and the Standard Output/Error of the nodes for info now.

jpfeuffer commented 4 years ago

Just a random guess: did you configure the Input database in the ID Metanode?

pavel-shliaha commented 4 years ago

I have installed:

  1. KNIME
  2. OpenMS-2.5.0-Win64.exe from https://www.openms.de/download/openms-binaries
  3. OpenMS extension through KNIME
  4. OpenMS-2.5-prerequisites-installer.exe

the workflow still does not run. Should I install anything else? This is the error message.

(or a complete workflow) generated with an older version of the tool. If you do not reconfigure the node (marked with an exclamation mark), the current defaults will be loaded instead.

ERROR LoadWorkflowRunnable Errors during load: Status: Error: Identification_quantification_isobaric_MSstatsTMT 0 loaded with errors ERROR LoadWorkflowRunnable Status: Error: Identification_quantification_isobaric_MSstatsTMT 0 ERROR LoadWorkflowRunnable Status: Error: MzTabExporter 0:227 ERROR LoadWorkflowRunnable Status: Error: Loading model settings failed: ERROR LoadWorkflowRunnable GenericKNIMENodes: ERROR LoadWorkflowRunnable Maybe you are loading node settings (or a complete workflow) generated with an older version of the tool. ERROR LoadWorkflowRunnable If you do not reconfigure the node (marked with an exclamation mark), ERROR LoadWorkflowRunnable the current defaults will be loaded instead. ERROR LoadWorkflowRunnable - Entry for parameter MzTabExporter.1.opt_columns not found in settings.xml. WARN IsobaricAnalyzer 0:235 Can't continue loop as the workflow was restored with the loop being partially executed. Reset loop start and execute entire loop again. WARN MSGFPlusAdapter 0:237:3 Can't continue loop as the workflow was restored with the loop being partially executed. Reset loop start and execute entire loop again. WARN IsobaricAnalyzer 0:235 Can't continue loop as the workflow was restored with the loop being partially executed. Reset loop start and execute entire loop again.

pavel-shliaha commented 4 years ago

I have reimported the workflow and it seems to be working

jpfeuffer commented 4 years ago

@oliveralka did you check: https://github.com/OpenMS/Tutorials/pull/166#issuecomment-529812404

oliveralka commented 4 years ago

@jpfeuffer Yes, checked some time ago - is set to precursor.

Note: The current workflow does not have the protein level anymore, since we have to fix the inference first and input it to the MSstatsConverter.

pavel-shliaha commented 4 years ago

Looks good. Minor changes. Then we can go over the workflow together via Skype.

Dear Jennifer and Oliver, could you please add me to the skype discussion as well? I use a lot of MS3 SPS and I think I could provide some valuable input to the discussion, as well as clarify some questions for myself. I wanted to ask about whether certain functionalities are implemented in openMS. In particular:

1) Could yo clarify why IDConflictResolver is used with isobaric mass tagging? 2) I understand that OpenMS workflow suggests using MSStats node for protein inference, but I actually wanted to export the ids with quant values and then look through the table myself. Is there a node that will convert IDConflictResolver output consensusXML to a csv output where the quantitation values are one line for PSM (wide format) or one line per channel (long format). I tried TextExporter, but it does not seem to generate this output 3) is there a node to estimate precursor contamination? 4) is there a node to estimate which of the fragments selected for MS3 originate from MS2 fragments which have been identified as true fragments from the identified peptide?

jpfeuffer commented 4 years ago

Hi Pavel,

sorry, the meeting is right now. But you will be in the data clinic tomorrow right?

1) It only makes sure that the top hit for the corresponding MS2 spectrum is retained (and not more). You could probably achieve the same with an IDFilter after the search engine. 2) The MSstats node just rearranges the tables to be compatible with MSstats. It does not perform any inference. TextExporter should export one line per PSM in the "FEATURE" section. The intensities should be in the columns intensity_0 - intensity_9 (or similar). An alternative would be the MzTabExporter (but I did not double-check the completeness of the output for TMT yet). 3) Yes, if you export meta values in the TextExporter, there should be a column with a precursor_purity score. 4) No, but that is a good idea.

jpfeuffer commented 4 years ago

@pavel-shliaha Regarding 4): How do you get the information about the isolated ions in the MS2. I have an SPS mzML here and all the MS3 spectra only report a single selected precursor ion:

              <selectedIonList count="1">
                <selectedIon>
                  ...

Not sure if this is a bug in the mzML converters (e.g. proteowizard) or if this information is just not available in the raw file.

pavel-shliaha commented 4 years ago

I am looking at the MSGFPlusAdapter and it does not have the TMTPro modification implemented. Could you please have a look?

jpfeuffer commented 4 years ago

Hi, which Unimod accession is this?

pavel-shliaha commented 4 years ago

accession number is 2016

oliveralka commented 4 years ago

I added the data here: https://abibuilder.informatik.uni-tuebingen.de/archive/openms/Tutorials/Data/isobaric_MSV000084264/

@jpfeuffer The path to the data has still to be added to the document, but please let me know what you think of the current version.

handout_isobaric_20200729.pdf